Non-Targeted Screening of Biosolids with the Xevo™ MRT Mass Spectrometer Reveals New Isoforms of PFAS

Importance of the topic

Per- and polyfluoroalkyl substances (PFAS) are environmentally persistent, bioaccumulative contaminants that occur in complex matrices such as biosolids, landfill leachate and surface water. Accurate, comprehensive characterisation of PFAS mixtures is essential for regulatory compliance, risk assessment and source control. Non-targeted screening (NTS) using high-resolution mass spectrometry (HRMS) complements targeted methods by revealing known, suspect and previously unrecognized PFAS species that contribute to total PFAS burden.

Goals and overview of the study

The study aimed to evaluate the capability of a benchtop multi-reflecting time-of-flight HRMS (Xevo MRT) paired with an integrated informatics platform (waters_connect, including UNIFI and Pattern Analysis applications) to:

• Quantify PFAS covered by U.S. EPA Method 1633 across biosolid, leachate and river water reference materials;
• Apply an NTS workflow to mine the same datasets for additional PFAS-like components and isoforms; and
• Demonstrate how advanced processing (Kendrick mass/Kendrick mass defect analysis, diagnostic fragment and neutral loss flags, and visualization) supports discovery and prioritization of unknown PFAS.

Methodology

Sample preparation followed the EPA 1633 protocol: solid-phase extraction (SPE) and clean-up from river water (250 mL), biosolid (0.5 g, NIST SRM 2781) and landfill leachate (25 mL, LGC 6177). River water was spiked with native and labelled standards because an initial screen showed no native PFAS; biosolid and leachate samples were spiked only with internal standards. Replicates were combined and, where indicated, diluted with methanol prior to LC-HRMS analysis.
Chromatography used an ACQUITY Premier LC configured with PFAS-reducing components and an ACQUITY Premier BEH C18 analytical column (2.1 × 100 mm, 1.7 µm) plus an Atlantis Premier BEH C18 AX isolator column. Mobile phases were water:methanol (95:5) with 2 mM ammonium acetate (A) and methanol with 2 mM ammonium acetate (B); flow 0.3 mL/min, injection 2 µL, column 35 °C, sample 20 °C.
Mass spectrometry used the Xevo MRT Mass Spectrometer in negative electrospray (ESI-) with data-independent acquisition (MSE) across m/z 50–1200 at 10 Hz. A dual-point lock mass (leucine enkephalin) ensured high mass precision. Typical source and acquisition settings included capillary 0.5 kV, cone 10 V, source 100 °C, desolvation 250 °C, low CE 6 V and high CE ramp 20–70 V.
Data processing combined targeted quantitation in the UNIFI Application (EPA 1633 compound list; 40 PFAS quantified, total 69 analytes including labelled standards) with a non-targeted Pattern Analysis workflow. NTS screening used diagnostic fragment/neutral loss flags, Kendrick mass and Kendrick mass defect (CF2-normalized), m/C (m/z per estimated carbon count) filtering and Bugsel/Kaufman visualization to prioritise PFAS-like features among ~10,000 detected components.

Used instrumentation

• Xevo MRT Mass Spectrometer (multi-reflecting time-of-flight HRMS)
• ACQUITY Premier LC System with PFAS kit
• ACQUITY Premier BEH C18 analytical column (1.7 µm, 2.1 × 100 mm)
• Atlantis Premier BEH C18 AX isolator column
• waters_connect Software Platform (UNIFI Application and Pattern Analysis Application)

Main results and discussion

Targeted quantitation: Across calibration and QC runs the instrument achieved root mean square (RMS) mass accuracy ≤ 0.59 ppm, demonstrating suitability for EPA 1633-compliant targeted analysis. Forty PFAS were quantified across the matrices with identification confidence levels assigned using established confidence scales (Schymanski/Charbonnet conventions). Some analytes were assigned lower confidence when diagnostic fragments from labelled internal standards could not be uniquely resolved.
Non-target discovery: Applying the Pattern Analysis workflow reduced the large feature list (~10,000 components in the biosolid extract) to 11 PFAS-like components that passed mass defect, m/C and fragment/neutral-loss criteria. Of these 11, three components shared the molecular formula C10HF21O3S (m/z 598.9238) and clustered around the mass defect region occupied by perfluorosulfonates (PFSA). One of these matched the retention time and diagnostic sulfonate fragments of authentic linear perfluorodecane sulfonic acid (PFDS). Two additional chromatographically resolved isomers eluted earlier or slightly later than linear PFDS; one of these produced characteristic SO3- and FSO3- fragments (supporting a branched PFSA assignment), while another lacked those diagnostic fragments and is interpreted as a sulfonate-ester-like species. Retention time separations and fragment patterns suggest at least two branched PFDS isoforms plus a sulfonate-ester isomer; annotation was supported by NIST PFAS library and PubChem formula searches but remains putative (confidence levels 3a/3d for branched PFDS isomers and 3c for the sulfonate-ester-like isomer).
Analytical implications: The study illustrates how high mass accuracy, MSE fragmentation and informed visualization/filtering substantially reduce candidate space and enable detection of isoforms that targeted lists miss. The lack of unique terminal-fragment signatures for some isomers underscores the importance of complementary MS/MS (data-dependent acquisition) or authentic standards to raise identification confidence.

Benefits and practical applications of the method

• Expanded detection space: HRMS NTS adds discovery power to targeted monitoring, improving estimates of total PFAS burden in complex matrices.
• Regulatory readiness: The platform met EPA 1633 analytical demands, supporting compliance workflows while enabling exploratory screening for non-regulated PFAS.
• Integrated informatics: Combining UNIFI and Pattern Analysis in a single environment improved traceability, reduced workflow gaps and simplified PFAS prioritization.
• Prioritization for follow-up: The approach identifies candidate unknowns for targeted follow-up (MS/MS, synthesis of standards, or toxicity assessment), enabling efficient allocation of confirmatory resources.

Future trends and possibilities for application

• Broader spectral libraries and curated PFAS databases will accelerate confident annotation of isoforms and transformation products.
• Routine incorporation of targeted data-dependent acquisition (DDA) or enhanced fragmentation strategies and orthogonal separations (e.g., ion mobility) will improve structural elucidation of branched and esterified PFAS.
• Synthesis and availability of authentic standards for branched isoforms and sulfonate esters will be critical to convert tentative annotations into confirmed identifications and quantitative calibrations.
• Integration of fluorine mass-balance approaches and total organofluorine screening will complement NTS to estimate unknown PFAS load.
• Standardized NTS workflows and harmonized confidence reporting will support regulatory uptake and inter-laboratory comparability.

Conclusion

This work demonstrates that a high-resolution multi-reflecting TOF platform combined with a comprehensive informatics environment can deliver both EPA 1633-grade targeted quantitation and effective non-targeted discovery of PFAS in complex environmental matrices. High mass accuracy, MSE fragmentation and Kendrick/KMD-based filtering enabled detection of multiple PFDS isoforms and other PFAS-like features that would be missed by targeted-only methods. Moving from tentative to confirmed identification will require targeted MS/MS acquisition and authentic standards, but the presented workflow provides a pragmatic pipeline for prioritising unknown PFAS for follow-up.

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